Probiotic potential of Saccharomyces cerevisiae GILA with alleviating intestinal inflammation in a dextran sulfate sodium induced colitis mouse model

Recently, several probiotic products have been developed; however, most probiotic applications focused on prokaryotic bacteria whereas eukaryotic probiotics have received little attention. Saccharomyces cerevisiae yeast strains are eukaryotes notable for their fermentation and functional food applications. The present study investigated the novel yeast strains isolated from Korean fermented beverages and examined their potential probiotic characteristics. We investigated seven strains among 100 isolates with probiotic characteristics further. The strains have capabilities such as auto-aggregation tendency, co-aggregation with a pathogen, hydrophobicity with n-hexadecane,1,1-diphenyl-2-picrylhydrazyl scavenging effect, survival in simulated gastrointestinal tract conditions and the adhesion ability of the strains to the Caco-2 cells. Furthermore, all the strains contained high cell wall glucan content, a polysaccharide with immunological effects. Internal transcribed spacer sequencing identified the Saccharomyces strains selected in the present study as probiotics. To examine the effects of alleviating inflammation in cells, nitric oxide generation in raw 264.7 cells with S. cerevisiae showed that S. cerevisiae GILA could be a potential probiotic strain able to alleviate inflammation. Three probiotics of S. cerevisiae GILA strains were chosen by in vivo screening with a dextran sulfate sodium-induced colitis murine model. In particular, GILA 118 down-regulates neutrophil–lymphocyte ratio and myeloperoxidase in mice treated with DSS. The expression levels of genes encoding tight junction proteins in the colon were upregulated, cytokine interleukin-10 was significantly increased, and tumor necrosis factor-α was reduced in the serum.


Aggregation and adhesion properties to caco-2 cell. Coaggregation with pathogen was high in S.
boulardii CNCM I-745. We selected S. cerevisiae GILA stains with more significant or similar to coaggregation ability than S. boulardii CNCM I-745 ( Table 2). Most of the isolated yeasts showed high autoaggregation properties. Aggregation characteristics of yeast are related to sporulation 21 . Notably, 80% autoaggregation after 24 h of incubation was based on colony formation 22 , which affects host colonization after entry. Those strains with autoaggregation ability could also have coaggregation ability with the pathogen. S. boulardii CNCM I-745 had between 60 and 85% coaggregation with the three pathogens, Staphylococcus aureus ATCC 25922, Enterococcus faecalis ATCC 29212 and Escherichia coli K88.
Adhesion assay to Caco-2 cell show all S. cerevisiae GILA strain's adhesion ability (4% to 15%). When compared with the control yeast strain, S. boulardii CNCM I-745, S. cerevisiae GILA 100, 118, 137, 197 were not significantly (p > 0.05) different. S. cerevisiae GILA 106 was significantly (p < 0.05) lower than GILA 100 (Fig. 2).   Alleviating inflammation in 264.7 cells and splenocyte. NO is related to various immunological procedures such as host defense, immunoregulation and signal transduction and are importance mediators triggering gastrointestinal disease 23 . NO is produced from L-arginine by an enzyme of nitric oxide synthase (NOS) and the inducible isoform of NO (iNOS) during inflammation where iNOS is activated by pro-inflammatory cytokines like tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6). For examining the effects of selected S. cerevisiae GILA, 10 ng/ml of LPS was treated in 264.7 cells for 48 h to induce inflammation and NO production. Selected S. cerevisiae GILA significantly (p < 0.001) suppressed NO production induced by LPS compared with the positive control treated LPS only. S. boulardii CNCM I-745 has shown anti-inflammatory effects compared with the LPS treatment group (Fig. 5).
Alleviating the intestinal inflammation in a dextran sulfate sodium-induced colitis mouse model. During DSS treatment with yeast period, the S. cerevisiae GILA115 group showed body weight loss more than the normal group (p < 0.01) on day11. In contrast the other S. cerevisiae GILA groups were not significantly (p > 0.05) different (Fig. 6a). Stool consistency and bleeding score were significantly lower in the S. cerevisiae GILA 59, 100, 118, and 137 groups than in the DSS group, although S. cerevisiae GILA 59 and 100 groups lose more weight (8-10%) than S. cerevisiae GILA 118 and 137 groups (4-8%). Consequently, S. cerevisiae GILA 118 and 137 groups were significantly (p < 0.05) lower than the DSS treatment group when compared with the disease activity index (DAI) score (Fig. 6b). The relative colon length rate was not significantly different ( Fig. 6c,d) whereas the relative spleen weight rate was significantly different between the normal and DSS treatment groups (Fig. 6e,f). S. cerevisiae GILA100 and 118 groups were similar spleen weight rates to the normal group (Fig. 6e). Standard scores were calculated using normal and DSS group scores. The total score showed GILA 100, 118, and 137 groups were similar to the normal group than the other groups (Fig. 6g).
To investigate the therapeutic properties of S. cerevisiae GILA strains in vivo, the DSS group showed IBD-colitis symptoms, including increased neutrophil count, neutrophil-lymphocyte ratio (NLR) in blood, myeloperoxidase (MPO) in feces (Fig. 7a), and proinflammatory cytokine (TNF-α) in serum (Fig. 7b). Stool consistency and bleeding score results were related to NLR from complete blood cell count (Fig. 7a) 24,25 . This finding suggested that the DSS-induced increase in neutrophils may affect other biomarkers and cytokines. Neutrophil expression in blood is one of the main features of colitis [26][27][28] . Further analysis was conducted to investigate the amelioration of intestinal inflammation. The gene expression levels of mucin-2 (Muc-2), zonula occludens-1 (ZO-1), occludin and epithelial cadherin (E-cadherin) significantly increased compared with those in the DSS group. (Fig. 7c).
We found that the S. cerevisiae GILA group had significantly decreased NLR in the blood (p < 0.01), MPO in feces (p < 0.001), and TNF-α in serum (p < 0.05, p < 0.01 and p < 0.001) (Fig. 7a,b). No significant changes were observed for IL-6 in serum. Meanwhile, IL-10 in serum significantly increased in the S. cerevisiae GILA 118 group compared with that in the other groups. The S. cerevisiae GILA 118 group also showed significantly increased serum IL-10 levels compared with the DSS group (Fig. 7b). These results suggested that S. cerevisiae GILA 118 effectively inhibits the biomarker of IBD and the expression of IL-10, thereby ameliorating colitis in mice.   Relative spleen weight rate compared with that of Normal (e) and DSS (f) group. (g) S.c GILA strain's in vivo screening total score. Statical significance is indicated as follows: *p < 0.05, **p < 0.01 and ***p < 0.001.

Discussion
In our investigation, we selected a probiotic candidate when we applied S. cerevisiae GILA, which had a high survival rate in low pH and bile conditions. These results indicate that yeast which had high resistance to harsh condition also had high survival in GIT simulation model. S. cerevisiae GILA has almost over 90.0% survival rate in GIT model. Tolerance to GI tract could be considered in vivo conditions. Comparing the viable counts in the murine gastrointestinal tract could be shown survival rate in vivo 29 . Survival was an important factor of probiotics, but safety was also studied for selected probiotic potential yeast. None of the selected S. cerevisiae GILA strain had hemolytic activity and biogenic amine production. Yeast is also known to have antibiotic resistance. Yeasts aggregate via their cell wall mannose 30 ; a prominent aggregation ability indicates ample mannose. S. cerevisiae GILA stain showed 90% hydrophobicity (data not shown). The adhesion ability for Caco-2 cells, such as microbial adhesion to mucosa model 31 was 4-15%. These results show that S. cerevisiae GILA stain possesses cell adhesion ability to be used in probiotic preparation. www.nature.com/scientificreports/ The DPPH scavenging effect measures the antioxidant capacity related to the cell wall β-glucan content. β-Glucan is a β-d-glucose polysaccharide group and a component of the yeast cell wall. Its structure is a long, β-(1,6)-branched, β-(1,3)-glucan 32 . β-Glucan has an excellent antioxidant capacity 33 ; however, this could not explain the results for cell wall β-glucan (Fig. S1). Yeast itself may then possess an intrinsic antioxidant ability 34 . Therefore, there should be another experiment to quantify cell wall β-glucan. β-Glucan is recognized by the receptors on the host's immune cells 35 , enhancing immune function resulting in anticancer and anti-inflammatory effects 35,36 . Furthermore, yeast β-Glucan benefits host health by protecting it against pathogens 37 . The content of β-Glucan was calculated from total glucan by subtracting α-glucan. A large amount of β-glucan in the yeast cell walls was comparable to a previous study 38 . The S. cerevisiae GILA strain had more than 36% β-glucan; this result could imply a probiotic potential (Fig. S1). Commercial probiotics, such as Lactobacillus rhamnosus GG have 5% β-glucan (data not shown), respectively. S. cerevisiae has more than triple the amount of β-glucan than these Lactobacillus strains. Moreover, the structure of β-glucan should also be considered in future research. Since β-glucan modulates cytokines in human blood, it should be confirmed whether all structures of β-glucan are beneficial to host health 39 . This result indicated that β-glucan benefits the host's health and immune system. A future probiotic approach could consider yeast's β-glucan characteristics. Our results revealed the quantity of β-glucan in the yeast cell walls. This method could be applied during probiotics-related yeast and β-glucan screening. The result was unexpected from the DPPH scavenging effect, but all experimental strains had more than 36% β-glucan, which is a sufficient level 38 .
The cell wall had more than 36% β-glucan, and nitric oxide production was significantly lower than the control. The β-glucan quantity could be edequate, but we need more evidence of related probiotic functionality. Previous studies have reported the prevention of inflammation by yeast fermentate 40 ; there are also reports on β-glucan-mediated induction of proinflammatory cytokines 41 . Given that the quantity of β-glucan did not influence this outcome, another factor, such as the probiotic properties of the S. cerevisiae GILA strain, may have been responsible for reducing in proinflammatory cytokines. The physiological impact of yeast probiotics against the host was determined by their ability to relieve oxidative stress measured by fecal MPO level 42 . Activated neutrophils release MPO, a marker of oxidative stress, and destroy epithelial cells 26 . As demonstrated by DPPH scavenging capacity of Saccharomyces cerevisiae GILA in vitro (Fig. 3), in vivo results also prove this ability to relieve oxidative stress (Fig. 7a). The S. cerevisiae strain was resistant to ETEC infection 17 , and S. cerevisiae cell wall glucan had an immune-modulatory effect, which could affect colitis reduction 13 . Spleen weight could be due to alleviating intestinal immune response 43 . Moreover, the inflammation biomarkers were similar to those in the CBC test-neutrophil lymphocyte rate results 28 (Fig. 7a,b). There was no significant difference in the relative expression rate of the Muc-2 gene between the normal and DSS groups (Fig. 7c). Recovery through a 6-days period (Fig. S2) is considered maintaining in the DSS group. In this study, an increase in Muc-2 gene expression was regarded as an improved capacity of epithelial protection 44 . As a result, similar to this experiment, colitis was alleviated by increasing Muc-2 expression in intestinal goblet cells 45 . An increase in Muc-2 gene expression is assumed to reduce colitis (Fig. 7c). Furthermore, we elucidated that anti-inflammatory cytokine IL-10 in serum was more upregulated by S. cerevisiae GILA 118 than other S. cerevisiae GILA strains. These findings warrant further experiments, especially S. cerevisiae GILA 118 structure study with the DSS-colitis model. For this reason, S. cerevisiae could be developed as a useful probiotic in the future. Nevertheless, more evidence as a potential gut microbiota modulator 46 is required for the S. cerevisiae GILA strain-related probiotics. Thus, further additional research and development are required to characterize these probiotic candidates' functionality.

Conclusion
We screened and selected seven S. cerevisiae strains as probiotic properties were similar to or higher than S. boulardii CNCM I-745. The strain S. cerevisiae GILA100, GILA118, and GILA137 met the criteria for a probiotic which had to alleviate inflammatory effect in a DSS-induced colitis mouse, especially S. cerevisiae GILA 118 administration increased IL-10 in serum and also alleviated intestinal inflammation in mice compared with S. cerevisiae GILA 100 and GILA137 (Fig. 8).

Materials and methods
Isolation and culture conditions. Eight samples of rice wine were obtained from Gangwon-do and three from Chungcheong-do, both in Korea. Ninety-two strains of yeast were from makgeolli, and eight strains were from dongdongju. All isolates were confirmed by gram staining and cell morphology 47 .
Saccharomyces boulardii CNCM I-745, supplied by Jarrow Formulas (Los Angeles, USA), was used as a control for comparison with the isolated yeast strain. The yeast strains were incubated aerobically at 37℃ for 24 h before use simulating the conditions in a human host 48 . All broth and agar materials were obtained from Difco (USA).

Resistance to low pH and bile conditions.
To determine the ability of yeast strains to survive in GI conditions, the isolates were incubated in YPD broth at 37 °C for 24 h. Then, the cultured yeasts were centrifuged at 5500×g for 10 min at 4 °C. The pellets were incubated for 2 h in YPD broth, and adjusted to pH 2.0 with 1 N HCl. The sample (100 μL), diluted in phosphate-buffered saline (PBS), was spread on YPD agar according www.nature.com/scientificreports/ to the drop-plating method 49 . After incubation in YPD broth (pH 2.0) at 37 °C, the resistance of yeast to bile was estimated similarly. The pellets were incubated for 12 h in YPD broth with 3.0% bovine bile (Oxgall, Difco, USA). The survival rate at pH 2.0 and in 3.0% Oxgall was calculated using the following formula: acid and bile tolerance (%) = [yeast after 24 h incubation (log cfu/mL)/yeast after 2 h incubation at pH 2.0 (log cfu/mL) and in 3.0% Oxgall (log cfu/mL)] × 100 50 .

1-Diphenyl-2-picrylhydrazyl (DPPH) scavenging effect. The DPPH scavenging assay was performed
to compare the antioxidant abilities of the yeast strains. The yeast pellets were harvested, washed twice, and resuspended in 1 mL PBS. The resulting suspensions (800 μL) were added to 1 mL DPPH solution (0.2 mM in 80% methanol) and mixed by vortexing, followed by incubation for 30 min in the dark. After the incubation, the solutions were centrifuged at 12,400×g for 5 min, and 300 μL of each sample was transferred to a 96-well plate to measure the absorbance at 517 nm. The reconstitution of the standard was performed by adding ascorbic acid to 80% (v/v) methanol at a concentration of 1 mg/mL to 400 μL/mL. Five twofold serial dilutions were performed, and 80% methanol served as the zero standard 54 .  Table 3. All materials were obtained from Sigma-Aldrich (USA) or Difco (USA). The inorganic and organic solutions are mixed with distilled water. The pH of the juices and incubation time are adjusted to human physiological traits with a minor modification 55,56 . 7 ml of the S. boulardii CNCM I-745 and S. cerevisiae GILA strain were centrifuged, then resuspended in 1 ml PBS. Saliva was added and incubated for 5 min. After the incubation, gastric juice was mixed and incubated for 2 h. 12 ml of duodenum juice with 6 ml of bile juice were mixed and incubated for 2 and 5 h with agitation (60×g) at 37 °C. Yeast samples were harvested three times and serially diluted and plated onto YPD agar.
Adhesion assay. The Caco-2 cell line, obtained from the Korea cell line bank (KCLB, Seoul, Korea), was used between passages 40-60 for all experiments. The cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; corning, USA) supplemented with 10% heat inactivated FBS, 2 mM l-glutamine ml −1 , 100 U penicillin ml −1 , and 100 µg, streptomycin ml −1 at 37 °C in a 5% CO 2 atmosphere. After harvesting yeast, overnight cultures of yeast strains were suspended with preheated fresh DMEM media and adjusted to O.D 600 nm at 2.0 density (approximately 1 × 10 7 cfu ml −1 ). One millimeter of each yeast was inoculated to 12-well plates and incubated for 2 h at 37 in a 5% CO 2 atmosphere. Then, non-adherent yeasts were removed by washing with PBS twice, and the Caco-2 cells and attached yeast were lysed with 1 ml of 0.05% Trypsin-EDTA (Gibco, USA). The adherent yeast was enumerated by diluting the solution serially (1:10) with PBS from the initial and using the drop-plating method on YPD agar 4 .
Hemolytic activity and biogenic amine production. Safety assessments were conducted by measuring the hemolytic activity and biogenic amine production. The hemolytic activity was evaluated using blood agar plates supplemented with 5% (v/v) defibrinated sheep blood (KisanBio, Korea). The appearance of clear zones around the colonies confirmed by β-hemolysis. After the colony of each strain was streaked on the blood agar, the plates were incubated aerobically at 37 °C for 48 h 57 .
Biogenic amine production was analyzed according to the method of Bover-Cid and Hozapfel 58 . The isolates were streaked on decarboxylase media and incubated aerobically at 37 °C for 4 days. Decarboxylase activity was detected by the color change from yellow to blue.

ITS region sequencing and phylogenetic analysis.
Single colonies were submitted to SolGent Corporation (South Korea) for ITS sequencing. DNA extraction was performed using a boiling method by Chelex bead. The screened and selected strains were identified using amplified internal transcribed spacer ITS 1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS 4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) sequencing 59 . The polymerase-chain-reaction (PCR) reaction was performed in a BigDye® Terminatorv3.1 cycle sequencing kits. Sequencing was analyzed by ABI 3730XL DNA Analyzer (50 cm capillary). The primary measurement (identity, %) was compared to the yeast strain. Sequences were aligned by the NCBI GenBank database using the BLASTn. Phylogenetic analysis proceeded with MEGA software version 11 with neighbor-joining analysis Cell wall β-glucan. β-Glucan (%, w/w) was measured using the yeast glucan assay kit (Megazyme, Ireland).
Before the calculation of β-glucan, the yeast cell wall was autolyzed and hydrolyzed following the procedures of Pengkumsri et al. 38 . Briefly, yeast cells were incubated in pH 5.0 water at 50 °C for 48 h with shaking at 160 × g, then at 80 °C for 15 min in a water bath. After incubation, yeast cells were harvested by centrifugation for 10 min at 4 °C at 6900×g. The autolyzed yeast cells were mixed with 1.0 M NaOH/HCl and incubated at 80 °C with a stirrer for 2 h. Finally, the hydrolyzed cells' β-glucan content (%, w/w) was calculated following the assay kit protocol.

Statistical analyses.
The results are the means ± standard deviations of triplicate analyses. Pearson's correlation and Duncan's test were performed with SPSS (version 18.0), and results were analyzed to ANOVA using the GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Table 4. Gene primer sequences. www.nature.com/scientificreports/ Research involving animal participants. For the in vivo experiment, this study was carried out in accordance with the guidelines by the Korean Association for Laboratory Animals, and the protocol was approved by the Institutional Animal Care and Use Committee of Seoul National University (Approval No. SNU-200706-6-2). All studies were performed in compliance with the ARRIVE guidelines.

Data availability
The datasets generated during the current study are available in the GenBank (Web link: https:// www. ncbi. nlm. nih. gov/ genba nk/) repository, accession number: OQ247983, OQ247986, OQ248002, OQ248003, OQ248004, OQ248507, and OQ253417. Cell wall β-glucan content of selected S. cerevisiae strains and an overview of in vivo screening are available in Supplementary Figs. 1 and 2.